Enzyme immobilization has been investigated under the aspects of enabling recycling of enzymes used in chemical synthesis, of convenience of handling (e.g., separation from the product), and of enhanced stability of immobilized enzyme preparations. See, e.g., Sheldon (2007) Adv. Synth. Catal. 349:1289-1307. Accordingly, several methods of immobilizing enzymes have been described in the art, including coupling of enzymes to solid particles, crosslinking enzymes, encapsidizing enzymes, and immobilizing enzymes in solid matrices. See, e.g., Methods in Biotechnology: Immobilization of Enzymes and Cells (Jose M. Guisan ed., Humana Press 2nd ed. 2006). For cross-linking, enzymes can be crystallized, and then cross-linked to produce cross-linked enzyme crystals (CLECs). Alternatively, enzymes can be precipitated, and the precipitates can be treated to produce cross-linked enzyme aggregates (CLEAs®; see, Sheldon (2007), supra). As a crosslinking agent, glutaraldehyde is generally the agent of choice. However, glutaraldehyde has a disadvantage that many enzymes are inactivated during cross-linking, which is attributed to the small size of glutaraldehyde molecules and which is thought to penetrate into protein molecules and thereby inactivate amino acids crucial for enzyme activity (see, id.). Accordingly, dextran polyaldehyde was proposed as alternative cross-linking agent, with improvements as compared to glutaraldehyde shown for nitrilases and penicillin G acylase. See. Mateo et al. (2004) Biotech. Bioeng. 86:273-276.
Diagnostic test elements usually are manufactured for use in near-patient applications. Therefore, test elements must be robust with respect to handling and storing. This applies, in particular, to the test chemistry of the test elements. See, e.g., Hones et al. (2008) Diabetes Technol. Ther. 10:S10-S26.
Many diagnostic test elements, however, are based upon rather complex enzyme test chemistries present on the test elements. In particular, test elements can include a carrier and a detection layer, where the detection layer usually contains enzymes. It is decisive for proper functioning of the test elements that the enzymes remain biologically active during storing and upon treating. Since calibration for an individual measurement is usually not possible, the test elements are normally calibrated batch-wise. The calibration information for a batch of test elements is then stored and used for each test element of the batch regardless of individual differences in treating and storing.
However, pretreatments of diagnostic test elements and storage conditions can severely affect enzyme activity. For example, heat treatment, either during manufacturing or during storing of the test elements, can denature the enzymes so that the overall enzymatic activity of a test element is significantly reduced that, in turn, will result in erroneous test results when such a test element is used. Similarly, with increasing water content in the detection layer, enzymes and other components of the detection layer will tend to diffuse, which is a particular problem in test elements having more than one layer of test chemistry. This problem is particularly pronounced in applications where test elements are exposed to environmental conditions for an extended time such as, for example, in “open vial” applications or where test elements are provided in cartridges. Accordingly, enzymes used in such test elements have been absorbed to charged particles such as Transpafil. However, using charged particles for immobilization has the disadvantage of severely diluting the specific activity of the enzyme immobilized, as calculated on a per mass basis, which, in turn, necessitates an increase of layer thickness to provide the required enzyme activity per area. This increase in layer thickness, however, usually leads to increased reaction times due to slowed diffusion of the analyte into the layer, meaning that increased test times are required.
Accordingly, there is a need for means and methods of improving immobilization of polypeptide molecules. In particular, methods of immobilizing enzymes avoiding strong dilution of the specific activity per mass and/or for immobilizing enzymes in the form of finely dispersible aggregates are needed.